Genes constitute only a small proportion of the total mammalian genome, and the precise control of their expression in the presence of an overwhelming background of noncoding deoxyribonucleic acid (DNA) presents a substantial problem for their regulation. Noncoding DNA, containing introns, repetitive elements, and potentially active transposable elements requires effective mechanisms for its long term silencing. Mammals appear to have taken advantage of the possibilities afforded by cytosine methylation to provide a heritable mechanism for altering DNA-protein interactions to assist in such silencing. DNA methylation is essential for the development of mammals and plays a potential role during aging and cancer. The involvement of methylation in the regulation of gene expression and as an epigenetic modification marking imprinted genes is well established. In mammals, methylation occurs only at cytosine residues and more specifically only on cytosine residues adjacent to a guanosine residue, i.e. at the sequence CG. The detection and mapping of DNA methylation sites are essential steps towards understanding the molecular signals which indicate whether a given sequence is methylated.
This is currently accomplished by the so-called bisulfite method described by Frommer, M., et al., Proc. Natl. Acad. Sci. USA 89 (1992) 1827-1831, for the detection of 5-methyl-cytosines. The bisulfite method of mapping 5-methylcytosine uses the effect that sodium hydrogen sulfite reacts with cytosine but not or only poorly with 5-methyl-cytosine. Cytosine reacts with bisulfite to form a sulfonated cytosine reaction intermediate being prone to deamination resulting in a sulfonated uracil which can be desulfonated to uracil under alkaline conditions (see FIG. 1). It is common knowledge that uracil has the base pairing behavior of thymine different to the educt cytosine whereas 5-methylcytosine has the base pairing behavior of cytosine. This makes the discrimination of methylated or non-methylated cytosines possible by e.g. bisulfite genomic sequencing (Grigg, G., and Clark, S., Bioessays 16 (1994) 431-436; Grigg, G. W., DNA Seq. 6 (1996) 189-198) or methylation specific PCR (MSP) disclosed in U.S. Pat. No. 5,786,146. Basic studies on the reaction of uracil and cytosine derivatives with bisulfite have been performed by Shapiro et al., JACS 92 (1970)422-424.
There are various documents addressing specific aspects of the bisulfite reaction.
Hayatsu, H., et al., Biochemistry 9 (1970) 2858-2865 reacted uracil, cytosine or their derivatives with 1 M bisulfite, at a pH value around 6, at 37° C. for 24 hours. Hayatsu, H., et al., J. Am. Chem. Soc. 92 (1970) 724-726 describe the reaction of cytosine with 3 M bisulfite at a pH value around 6 at a temperature of 80° C. for 30 min. Slae and Shapiro, J. Org. Chem. 43 (1978) 4197-4200 describe the deamination of cytidine with 1 M bisulfite around neutral pH at various temperatures whereby the reaction time is not described. There were no investigations of the deamination of cytosine or methyl-cytosine in nucleic acids in these documents.
Paulin, R., et al., Nucl. Acids Res. 26 (1998) 5009-5010 investigate the effects of urea on the efficiency of bisulfite-mediated sequencing of 5-methylcytosine in DNA. The DNA is reacted with 3.44 M bisulfite in the presence of 5.36 M urea and 0.5 mM hydroquinone, at a pH value of 5.0 at a temperature of 55° C. for 15 hours.
Raizis, A. M., et al., Anal. Biochem. 226 (1995) 161-166 disclose a bisulfite method for 5-methylcytosine mapping that minimizes template degradation. They investigate a method minimizing template degradation using 5 M bisulfite solutions in the presence of 100 mM hydroquinone at a pH value of 5 at 50° C. A maximum yield of PCR product was observed after 4 hours. Other conditions as increased pH and lower temperatures were also investigated.
Grunau, C., et al., Nucleic Acids Res 29 (2001) e65-5, page 1 to 7, perform a systematic investigation of critical experimental parameters of the bisulfite reaction. They investigate bisulfite solutions of 3.87 to 4.26 and 5.2 to 5.69 M at a pH value of 5. Temperatures that were tested are 15, 35, 55, 80, 85 and 95° C. for 1, 4 and 18 hours. DNA degradation is a problem in these investigations.
Wang, R. Y., et al., Nucleic Acids Res. 8 (1980) 4777-4790 disclose the use of a 3 M bisulfite solution at a pH value of 5.5 at a temperature of 37° C. for various time periods in the bisulfite treatment of DNA. Feil, R., et al., Nucleic Acids Res 22 (1994) 695-696 disclose the use of a 3.5 M bisulfite solution at a pH value of 5 at a temperature of 0° C. for 24 hours in the bisulfite treatment of DNA. Clark, S. J., et al., Nucleic Acids Res 22 (1994) 2990-2997, disclose the use of a 3 to 4 M bisulfite solution at a pH value of 4.8 to 5.8 at a temperature of 37 to 72° C. for 8 to 16 hours in the bisulfite treatment of DNA. Tasheva, E. S., and Roufa, D. J., Mol. Cell. Biol. 14 (1994) 5636-5644 disclose the use of a 1 M bisulfite solution at a pH value of 5 at a temperature of 50° C. for 48 hours in the bisulfite treatment of fragments of genomic DNA. Grigg, G. W., DNA Seq 6 (1996) 189-198 discloses the use of a 3.1 M bisulfite solution at a pH value of 5 at a temperature of 50° C. for 16 hours in the bisulfite treatment of DNA. Komiyama, M., and Oshima, S., Tetrahedron Letters 35 (1994) 8185-8188 disclose the use of a 1 M bisulfite solution at a pH value of 5 at a temperature of 37° C. for 4 hours in the bisulfite treatment of DNA whereby diethylenetriamine is present.
Olek, A., et al., Nucleic Acids Res. 24 (1996) 5064-5066 disclose a method for bisulfite base sequencing whereby bisulfite treatment and subsequent PCR steps are performed on material embedded in agarose beads. A 5 M bisulfite solution at a pH value of 5 at a temperature of 50° C. is used for 4 hours in the bisulfite treatment of DNA.
A review of DNA methylation analysis can be found in Oakeley, E. J., Pharmacol. Ther. 84 (1999) 389-400.
Different additional components in the bisulfite mixture are disclosed by WO 01/98528, WO02/31186 or by Paulin, R., et al., Nucleic Acids Res 26 (1998) 5009-5010.
Kits for performing bisulfite treatments are commercially available from Intergen, now distributed by Serologicals Corporation, Norcross, Ga., USA, e.g. CpGenome™ DNA modification kit (http://www.serologicals.com/products/int_prod/index.html).
All prior art methods for the bisulfite treatment have disadvantages. Therefore, the problem to be solved by the present invention was to provide a method which overcomes the disadvantages of the prior art methods.